Compositions and methods for increasing the serum half-life of a therapeutic agent targeting complement c5

ABSTRACT

The disclosure features compositions and methods for increasing the half-life of a therapeutic agent (e.g., a C5 antagonist) in the serum of a subject (e.g., a human). Also featured are compositions and methods for: (i) decreasing the frequency by which a therapeutically effective amount of a therapeutic agent must be administered to a human having, suspected of having, or at risk for developing, a medical condition for which the therapeutic agent is effective and (ii) decreasing the dosage of the therapeutic agent required for therapeutic efficacy in a human having, suspected of having, or at risk for developing, a medical condition for which the therapeutic agent is effective. The methods include reducing the serum concentration of the antigen to which the therapeutic agent binds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application Ser. No. 61/806,687, filed Mar. 29, 2013, the entirecontent of which is hereby incorporated by reference.

TECHNICAL FIELD

The field of the invention is medicine, immunology, molecular biology,and protein chemistry.

BACKGROUND

The complement system acts in conjunction with other immunologicalsystems of the body to defend against intrusion of cellular and viralpathogens. There are at least 25 complement proteins, which are found asa complex collection of plasma proteins and membrane cofactors. Theplasma proteins make up about 10% of the globulins in vertebrate serum.Complement components achieve their immune defensive functions byinteracting in a series of intricate but precise enzymatic cleavage andmembrane binding events. The resulting complement cascade leads to theproduction of products with opsonic, immunoregulatory, and lyticfunctions. A concise summary of the biologic activities associated withcomplement activation is provided, for example, in The Merck Manual,16^(th) Edition.

The complement cascade can progress via the classical pathway (CP), thelectin pathway, or the alternative pathway (AP). The lectin pathway istypically initiated with binding of mannose-binding lectin (MBL) to highmannose substrates. The AP can be antibody independent, and can beinitiated by certain molecules on pathogen surfaces. The CP is typicallyinitiated by antibody recognition of, and binding to, an antigenic siteon a target cell. These pathways converge at the C3 convertase—the pointwhere complement component C3 is cleaved by an active protease to yieldC3a and C3b.

The AP C3 convertase is initiated by the spontaneous hydrolysis ofcomplement component C3, which is abundant in the plasma fraction ofblood. This process, also known as “tickover,” occurs through thespontaneous cleavage of a thioester bond in C3 to form C3i or C3(H₂O).Tickover is facilitated by the presence of surfaces that support thebinding of activated C3 and/or have neutral or positive chargecharacteristics (e.g., bacterial cell surfaces). This formation ofC3(H₂O) allows for the binding of plasma protein Factor B, which in turnallows Factor D to cleave Factor B into Ba and Bb. The Bb fragmentremains bound to C3 to form a complex containing C3(H₂O)Bb—the“fluid-phase” or “initiation” C3 convertase. Although only produced insmall amounts, the fluid-phase C3 convertase can cleave multiple C3proteins into C3a and C3b and results in the generation of C3b and itssubsequent covalent binding to a surface (e.g., a bacterial surface).Factor B bound to the surface-bound C3b is cleaved by Factor D to thusform the surface-bound AP C3 convertase complex containing C3b,Bb. (See,e.g., Müller-Eberhard (1988) Ann Rev Biochem 57:321-347.)

The AP C5 convertase—(C3b)₂,Bb—is formed upon addition of a second C3bmonomer to the AP C3 convertase. (See, e.g., Medicus et al. (1976) J ExpMed 144:1076-1093 and Fearon et al. (1975) J Exp Med 142:856-863.) Therole of the second C3b molecule is to bind C5 and present it forcleavage by Bb. (See, e.g., Isenman et al. (1980) J Immunol124:326-331.) The AP C3 and C5 convertases are stabilized by theaddition of the trimeric protein properdin as described in, e.g.,Medicus et al. (1976), supra. However, properdin binding is not requiredto form a functioning alternative pathway C3 or C5 convertase. (See,e.g., Schreiber et al. (1978) Proc Natl Acad Sci USA 75: 3948-3952 andSissons et al. (1980) Proc Natl Acad Sci USA 77: 559-562.)

The CP C3 convertase is formed upon interaction of complement componentC1, which is a complex of C1q, C1r, and C1s, with an antibody that isbound to a target antigen (e.g., a microbial antigen). The binding ofthe C1q portion of C1 to the antibody-antigen complex causes aconformational change in C1 that activates C1r. Active C1r then cleavesthe C1-associated C1s to thereby generate an active serine protease.Active C1s cleaves complement component C4 into C4b and C4a. Like C3b,the newly generated C4b fragment contains a highly reactive thiol thatreadily forms amide or ester bonds with suitable molecules on a targetsurface (e.g., a microbial cell surface). C1s also cleaves complementcomponent C2 into C2b and C2a. The complex formed by C4b and C2a is theCP C3 convertase, which is capable of processing C3 into C3a and C3b.The CP C5 convertase—C4b,C2a,C3b—is formed upon addition of a C3bmonomer to the CP C3 convertase. (See, e.g., Müller-Eberhard (1988),supra and Cooper et al. (1970) J Exp Med 132:775-793.)

In addition to its role in C3 and C5 convertases, C3b also functions asan opsonin through its interaction with complement receptors present onthe surfaces of antigen-presenting cells such as macrophages anddendritic cells. The opsonic function of C3b is generally considered tobe one of the most important anti-infective functions of the complementsystem. Patients with genetic lesions that block C3b function are proneto infection by a broad variety of pathogenic organisms, while patientswith lesions later in the complement cascade sequence, i.e., patientswith lesions that block C5 functions, are found to be more prone only toNeisseria infection, and then only somewhat more prone.

The AP and CP C5 convertases cleave C5 into C5a and C5b. Cleavage of C5releases C5a, a potent anaphylatoxin and chemotactic factor, and C5b,which allows for the formation of the lytic terminal complement complex,C5b-9. C5b combines with C6, C7, and C8 to form the C5b-8 complex at thesurface of the target cell. Upon binding of several C9 molecules, themembrane attack complex (MAC, C5b-9, terminal complement complex—TCC) isformed. When sufficient numbers of MACs insert into target cellmembranes the openings they create (MAC pores) mediate rapid osmoticlysis of the target cells.

While a properly functioning complement system provides a robust defenseagainst infecting microbes, inappropriate regulation or activation ofthe complement pathways has been implicated in the pathogenesis of avariety of disorders including, e.g., rheumatoid arthritis (RA); lupusnephritis; asthma; ischemia-reperfusion injury; atypical hemolyticuremic syndrome (aHUS); dense deposit disease (DDD); paroxysmalnocturnal hemoglobinuria (PNH); macular degeneration (e.g., age-relatedmacular degeneration (AMD)); hemolysis, elevated liver enzymes, and lowplatelets (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP);spontaneous fetal loss; Pauci-immune vasculitis; epidermolysis bullosa;recurrent fetal loss; multiple sclerosis (MS); traumatic brain injury;and injury resulting from myocardial infarction, cardiopulmonary bypassand hemodialysis. (See, e.g., Holers et al. (2008) Immunological Reviews223:300-316.) The down-regulation of complement activation has beendemonstrated to be effective in treating several disease indications ina variety of animal models. See, e.g., Rother t al. (2007) NatureBiotechnology 25(11):1256-1264; Wang et al. (1996) Proc. Natl. Acad.Sci. USA 93:8563-8568; Wang et al. (1995) Proc. Natl. Acad. Sci. USA92:8955-8959; Rinder et al. (1995) J. Clin. Invest. 96:1564-1572;Kroshus et al. (1995) Transplantation 60:1194-1202; Homeister et al.(1993) J. Immunol. 150:1055-1064; Weisman et al. (1990) Science249:146-151; Amsterdam et al. (1995) Am. J. Physiol. 268:H448-H457; andRabinovici et al. (1992) J Immunol 149:1744 1750.

SUMMARY

The present disclosure relates to compositions and methods forprolonging the half-life of a therapeutic agent—e.g., a C5 antagonistsuch as an anti-C5 antibody—in the serum of a subject (e.g., a human).The inventors appreciated that antigen-mediated clearance (i.e.,C5-driven clearance) contributes significantly to reducing the serumhalf-life of an antagonist anti-C5 antibody, such as eculizumab. Whileanti-C5 antibodies are highly effective at inhibiting complement invitro and in vivo (see, e.g., Hillmen et al. (2004) N Engl J Med350(6):552), the antibodies are particularly susceptible totarget-mediated clearance because of the high concentration of C5 inblood (see International application publication no. WO 2010/151526).The concentration of C5 protein in human serum is approximately 75 μg/mL(0.4 μM) (Rawal and Pangburn (2001) J Immunol 166(4):2635-2642) and theprotein is rapidly turned over. In fact, the half-life of human C5 inblood is approximately 63 hours. See Sissons et al. (1977) Clin Invest59:704-715. The abundance of C5 in serum requires a correspondingly highconcentration of a C5 antagonist (e.g., an anti-C5 antibody such aseculizumab) to effectively inhibit C5 in humans, e.g., humans afflictedwith a complement-associated disorder such as paroxysmal nocturnalhemoglobinuria (PNH). Yet, because a C5-bound anti-C5 antibody iseliminated at roughly the same rate as C5, as compared to the slowerclearance rate of an antibody expected in the absence ofantigen-mediated clearance, sustained inhibition of C5 and complementactivity in patients in need thereof also requires higher frequencyadministration of the antibody.

The inventors further appreciated that reducing the concentration of C5in the serum, and particularly administering a C5 antagonist to a humanin whom the concentration of C5 is reduced, provides at least twoadvantages: (a) reducing the required dosage amount of the C5 antagonistand/or (b) reducing the required dosage frequency of the C5 antagonist.A C5 antagonist is generally administered to patients afflicted withhemolytic disease in an amount necessary to maintain serum complementactivity below 20% of the complement activity in normal serum in theabsence of the C5 antagonist. For eculizumab, the concentration of theantibody required to maintain serum complement activity below this levelis approximately greater than or equal to 35 to 50 μg/mL (i.e.,approximately 1 mole of eculizumab per 2 moles of C5). See, e.g.,International patent application publication nos. WO 2005/074607 and WO2010/054403. Below the 20% threshold, complement activity issufficiently reduced to control, among other things, C5b-mediatedhemolysis in the patients. However, generally, if the complementactivity in such a patient exceeds that 20% threshold, the patient willexperience a breakthrough event. For a patient suffering from PNH, thisbreakthrough event is characterized by hemoglobinuria, dysphagia, andincreased risk for thrombosis. See, e.g., International patentapplication publication no. WO 2005/074607. For a patient suffering fromatypical hemolytic uremic syndrome (aHUS), breakthrough can be even moredevastating—thrombosis and kidney failure. See International patentapplication publication no. WO 2010/054403.

Reducing the concentration of C5 in the serum effectively reduces theconcentration of the C5 antagonist required to maintain serum complementactivity at below 20%. Thus, when the serum concentration of C5 isreduced, the dose amount of a C5 antagonist (e.g., an anti-C5 antibodysuch as eculizumab) can be less than the dose amount of the antagonistrequired for maintaining serum complement activity at below 20% when theserum C5 concentration is not reduced.

In addition or in the alternative, a lower level of C5 in the serum cansignificantly reduce the impact of C5 antigen-mediated clearance on anantagonist anti-C5 antibody administered to a human. Since the syntheticrate of C5 drives the rate at which a C5 antagonist such as eculizumabis cleared, a reduction in that synthetic rate by virtue of, e.g., ansiRNA that inhibits expression of C5, would correspondingly prolongserum residency of the C5 antagonist. Thus, a medical practitionertreating a patient afflicted with a complement-associated disorder canadminister a C5 antagonist to the patient less frequently and/or at alower dose and still achieve clinical efficacy for an equal or longerperiod of time. The ability to administer a lower dose of the C5antagonist, or the ability to administer the C5 antagonist lessfrequently, also allows for additional delivery routes such as, e.g.,subcutaneous administration or intramuscular administration andopportunities for self-administration by patients in their homes.

Accordingly, the disclosure provides a method for increasing thehalf-life of a therapeutic agent in the serum of a subject (e.g., ahuman). The method comprises: (i) administering to the subject acompound that reduces in the subject the serum concentration of theantigen (e.g., soluble antigen) to which a therapeutic agent binds tothereby reduce the concentration of the antigen in the serum of thehuman and (ii) administering to the subject the therapeutic agent,wherein a reduced antigen concentration in the serum of the subjectincreases the serum half-life of the therapeutic agent administered tothe subject. The therapeutic agent can be, e.g., an antibody orantigen-binding fragment thereof, a small molecule, an aptamer, or anon-antibody, scaffold protein.

In some embodiments, antigen to which the therapeutic agent binds is acomplement protein (e.g., a human protein) such as, e.g., C1, C4, C3,C2, C5, C6, C7, C8, C9, properdin, complement factor B, complementfactor D, MBL, MASP1, MASP2, or MASP3.

Also featured is a method for increasing the half-life of a C5antagonist (e.g., an anti-C5 antibody) in the serum of a human, whichmethod includes: (i) administering to the human a compound that reducesthe concentration of complement component C5 in the serum of the humanto thereby reduce the C5 concentration in the serum of the human and(ii) administering to the human the C5 antagonist, wherein a reduced C5concentration in the serum of the human increases the serum half-life ofthe C5 antagonist administered to the human.

As used herein, the term “C5 antagonist” or like terms means any agentthat binds to complement component C5 protein and inhibits the cleavageof C5 into fragments C5a and C5b by C5 convertase (e.g., an alternativepathway or classical pathway C5 convertase). As used herein, the term“C5 convertase” can refer to either the classical pathway C5 convertaseC4bC2aC3b or the alternative pathway convertase (C3b)₂Bb.

As noted above, administration of the C5 antagonist (e.g., an antagonistanti-C5 antibody) to the human in a context in which the serumconcentration of C5 is reduced below normal levels has severaladvantageous effects, e.g., for humans having, suspected of having, orat risk for developing, a complement-associated condition. For example,because there is less C5 antigen in the serum of the human, the amountof the C5 antagonist administered to the human having the reduced C5concentration can be less than the therapeutically effective amount ofthe C5 antagonist required without the reduction in C5 concentration(that is in the context of normal serum C5 concentration). In addition,or in the alternative, the frequency of administration of atherapeutically effective amount of the C5 antagonist to the humanhaving a reduced C5 concentration can be less often than the frequencyof administration of the therapeutically effective amount to the humanrequired without the reduction in C5 concentration. For example, insteadof once, biweekly dosing, the required dosing frequency could beextended to, e.g., once monthly, once bimonthly, or even once everythree months. Thus, reducing the serum C5 concentration can one or bothof: (a) reduce the amount of the dose of the C5 antagonist required and(b) the frequency of dosing of the C5 antagonist, and yet still achievethe same therapeutic effect in the human as a higher dose and/or morefrequent administration of the C5 antagonist required if the human had anormal serum C5 concentration.

Thus, in another aspect, the disclosure features a method for decreasingthe frequency by which a therapeutically effective amount of a C5antagonist must be administered to a human having, suspected of having,or at risk for developing, a complement-associated disorder. The methodincludes: (i) administering to the human a compound that reduces theconcentration of complement component C5 in the serum of the human tothereby reduce the C5 concentration in the serum of the human and (ii)administering to the human a therapeutically effective amount of the C5antagonist, wherein a reduced C5 concentration in the serum of the humandecreases the frequency by which a therapeutically effective amount of aC5 antagonist must be administered to the human.

Moreover, in another aspect, the disclosure features a method fordecreasing the dosage of a C5 antagonist required for therapeuticefficacy in a human having, suspected of having, or at risk fordeveloping, a complement-associated disorder. The method comprises: (i)administering to the human a compound that reduces the concentration ofcomplement component C5 in the serum of the human to thereby reduce theC5 concentration in the serum of the human and (ii) administering to thehuman a therapeutically effective amount of the C5 antagonist, wherein areduced C5 concentration in the serum of the human decreases the dosageof a C5 antagonist required for therapeutic efficacy in the human.

Also featured is a method for treating a subject (e.g., a human)afflicted with a complement-associated disorder. The method comprisesadministering to the subject a therapeutically-effective amount of anantagonist of complement component C5, wherein the subject has a reducedserum concentration of C5, relative to the normal serum concentration ofC5, as the result of the prior administration to the patient of acompound that reduces the serum concentration of C5. In someembodiments, the normal serum concentration of C5 is the average C5concentration of like healthy subjects of the same species. In someembodiments, the normal serum concentration of C5 is the serum C5concentration in the subject (e.g., human) prior to administration ofthe compound and subsequent reduction of C5 concentration.

The disclosure also provides a method for treating a subject afflictedwith a complement-associated disorder, which method comprisesadministering to the subject a compound that reduces the serumconcentration of C5 in the subject in an amount effective to reduce theserum concentration of C5 in the subject. The compound is also to beused therapeutically in conjunction with an inhibitor of C5, wherein, asa result of administration of the compound, the inhibitor of C5 can beadministered to the subject (e.g., a human) less frequently and/or inlower dose amounts than the frequency of administration and/or doseamount that would be administered if the subject had a normal serumconcentration of C5. Thus, the subject is one who is to be treated witha C5 inhibitor, and/or in need of treatment with the C5 inhibitor, aspart of the therapeutic strategy for treating the subject'scomplement-associated disorder.

Further disclosed is a method for treating a subject (e.g., a human)afflicted with a complement-associated disorder, which method comprisesadministering to the subject a compound that reduces the serumconcentration of complement component C5, wherein the subject is also tobe treated with a C5 antagonist.

In another aspect, the disclosure provides a composition (e.g., apharmaceutical composition) comprising a compound that reduces the serumconcentration of C5 in a subject (e.g., a human), the composition foruse in treating a subject afflicted with a complement-associatedcondition. The subject can be, e.g., one who is to be administered a C5antagonist (e.g., an anti-C5 antibody).

In another aspect, the disclosure features a composition (e.g., apharmaceutical composition) comprising a C5 antagonist (e.g., an anti-C5antibody) for use in treating a subject afflicted with acomplement-associated disorder. The subject can be one who has a reducedlevel of C5 expression as a result of the action of a compound (thatreduces expression of C5) administered to the subject.

In some embodiments of any of the methods described herein, the compoundreduces the level of expression by one or more cells in the subject(e.g., human) of the antigen to which the therapeutic agent binds. Thus,in some embodiments of any of the methods described herein, the compoundreduces the level of expression of human complement component C5 by oneor more cells in the human.

In some embodiments of any of the methods described herein, the compoundinhibits transcription of a gene encoding the antigen (e.g., a humancomplement component C5 gene) to which the therapeutic agent (e.g., a C5antagonist such as an anti-C5 antibody) binds. In some embodiments ofany of the methods described herein, the compound inhibits translationof an mRNA encoding the antigen to which the therapeutic agent binds. Insome embodiments of any of the methods described herein, the compoundreduces the stability of an mRNA encoding the antigen to which thetherapeutic agent binds. Thus, in some embodiments, e.g., in embodimentswhere the therapeutic agent is a C5 antagonist such as an anti-C5antibody, the compound inhibits transcription of a human complementcomponent C5 gene. In some embodiments, the compound inhibitstranslation of an mRNA encoding human complement component C5. In someembodiments, the compound reduces the stability of an mRNA encodinghuman complement component C5. In some embodiments, the compound is ansiRNA specific for the mRNA encoding the antigen to which thetherapeutic agent binds (e.g., an siRNA specific for human complementcomponent C5). In some embodiments, the compound is an antisense nucleicacid complementary to a mRNA encoding the antigen to which thetherapeutic agent binds (e.g., an antisense nucleic acid complementaryto a mRNA encoding human complement component C5).

In some embodiments of any of the methods described herein, the C5antagonist is selected from the group consisting of: MB12/22,MB12/22-RGD, ARC187, ARC1905, SSL7, and OmCI.

In some embodiments of any of the methods described herein, the C5antagonist is an antibody, or an antigen-binding fragment thereof, thatbinds to complement component C5 and inhibits the cleavage of C5 by theC5 convertase into fragments C5a and C5b. The antibody orantigen-binding fragment thereof can be, e.g., a polyclonal antibody, arecombinant antibody, a diabody, a chimerized or chimeric antibody, adeimmunized antibody, a fully human antibody, a single chain antibody, adomain antibody, an Fv fragment, an Fd fragment, an Fab fragment, anFab′ fragment, or an F(ab′)₂ fragment. In some embodiments, the antibodyor antigen-binding fragment thereof is a bispecific antibody orbispecific antigen-binding fragment. In some embodiments, the antibodyor antigen-binding fragment thereof can be a DVD-Ig antibody.

In some embodiments of any of the methods described herein, the antibodyis eculizumab. In some embodiments of any of the methods describedherein, the antigen-binding fragment of an anti-C5 antibody ispexelizumab.

In some embodiments of any of the methods described herein, the compoundreduces the serum concentration of the antigen (e.g., human C5) to whichthe therapeutic agent binds by at least 2 (e.g., at least 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, or 100) %. For example, in some embodiments of any of themethods described herein, the compound reduces the serum concentrationof C5 by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70%.

In some embodiments, the serum concentration of C5 is reduced to 40% orless (e.g., 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%,27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5%) of the normal serumconcentration of C5 (e.g., the average serum concentration of C5 amongindividuals or the serum C5 concentration in the serum of the patientprior to treatment with the compound).

In some embodiments, the compound is administered to the patient in anamount effective to reduce the expression of C5 protein and/or mRNA byliver cells by at least 20 (e.g., at least 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, or 95) %.

In some embodiments of any of the methods described herein, the compoundcan be chronically administered to the subject (e.g., the human). Insome embodiments of any of the methods described herein, the therapeuticagent (e.g., a C5 antagonist) can be chronically administered to thesubject (e.g., the human). In some embodiments of any of the methodsdescribed herein, both the compound (e.g., a nucleic acid compound thatreduces expression of C5) and the therapeutic agent (e.g., a C5antagonist) can be chronically administered to the subject (e.g., thehuman). As used herein, “chronically administered,” “chronic treatment,”“treating chronically,” or similar grammatical variations thereof referto a treatment regimen that is employed to maintain a certain thresholdconcentration of a compound or therapeutic agent in the blood, serum, orplasma of a patient required for activity in the patient over aprolonged period of time. Accordingly, a patient chronically treatedwith a compound can be treated for a period of time that is greater thanor equal to 2 weeks (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,or 52 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months; or 1, 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,10.5, or 12 years or for the remainder of the patient's life) with thecompound in an amount and with a dosing frequency that are sufficient tomaintain in the patient's blood a reduction in the concentration of theantigen to which the therapeutic agent binds. Similarly, a patientchronically treated with a C5 antagonist such as an anti-C5 antibody canbe treated for a period of time that is greater than or equal to 2 weeks(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks; 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months; or 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 12 years orfor the remainder of the patient's life) with the therapeutic agent inan amount and with a dosing frequency that are sufficient to maintaininhibition or substantial inhibition (less than 20% of the complementactivity present in a human in the absence of a C5 antagonist) ofsystemic complement activity in the patient. In some embodiments, thecomplement inhibitor can be chronically administered to a patient inneed thereof in an amount and with a frequency that are effective tomaintain serum hemolytic activity at less than or equal to 20 (e.g., 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or even below 5) %. See,e.g., Hill et al. (2005) Blood 106(7):2559. In some embodiments, thecomplement inhibitor can be administered to a patient in an amount andwith a frequency that are effective to maintain serum lactatedehydrogenase (LDH) levels at within at least 20 (e.g., 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or even below 5) % of the normalrange for LDH. See Hill et al. (2005) supra. In some embodiments, thecomplement inhibitor is administered to the patient in an amount andwith a frequency that are effective to maintain a serum LDH level lessthan 550 (e.g., less than 540, 530, 520, 510, 500, 490, 480, 470, 460,450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320,310, 300, 290, 280, or less than 270) IU/L. As noted above,administering a C5 antagonist (e.g., an anti-C5 antibody such aseculizumab) in the context of reduced serum C5 levels can reduce theamount (dose) of the C5 antagonist required for therapeutic efficacyand/or can reduce how often the same dose or a reduced dose must beadministered to the patient, and still maintain therapeutic complementinhibition.

In some embodiments of any of the methods described herein, the compoundis administered to the human at least once weekly for at least threeweeks. In some embodiments of any of the methods described herein, thecompound is administered to the human at least once weekly for at leastsix weeks. In some embodiments of any of the methods described herein,the compound is administered to the human at least once weekly for atleast six months.

In some embodiments of any of the methods described herein, the compoundis subcutaneously administered to the human. In some embodiments of anyof the methods described herein, the therapeutic agent (e.g., a C5antagonist) and the compound are administered to the human usingdifferent routes of administration. In some embodiments of any of themethods described herein, the therapeutic agent (e.g., a C5 antagonist)and the compound are administered to the human under different dosingschedules.

In some embodiments, each dose of the compound administered to thepatient is between 0.01 mg to 30 mg per kg weight of the patient. Insome embodiments, the dose is between 0.01 mg/kg and 35 mg/kg (e.g., 0.1mg/kg-10 mg/kg, 1 mg/kg-15 mg/kg, 0.1 mg/kg-5 mg/kg, 3 mg/kg-5 mg/kg, 10mg/kg-30 mg/kg, 15 mg/kg-35 mg/kg, 1 mg/kg-3 mg/kg, 10 mg/kg-20 mg/kg,0.01 mg/kg-1 mg/kg, 5 mg/kg-20 mg/kg, 5 mg/kg-15 mg/kg, or 2 mg/kg to 15mg/kg). In some embodiments, the dose of the compound is at least 0.01(e.g., 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, or 36) mg/kg. In some embodiments the dose of the compound is atleast 0.01 mg/kg (e.g., at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or10) mg/kg, but no greater than 50 (e.g., 49, 48, 47, 46, 45, 44, 43, 42,41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24,23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11) mg/kg.

In some embodiments, the serum half-life of the therapeutic agent (e.g.,the C5 antagonist such as an anti-C5 antibody) is increased by at least2 (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100) fold in thecontext of a reduced serum concentration of the antigen to which thetherapeutic agent binds. That is, in some embodiments of any of themethods described herein, the serum half-life of a C5 antagonist such asan anti-C5 antibody is increased by at least 2 (e.g., at least 3, 4, 5,6, 7, 8, 9, 10, 20, 50, or 100) fold in the context of a reduced serumconcentration of C5. For example, in some embodiments of any of themethods described herein, the serum half-life of a therapeutic agent canbe increased from, e.g., 3 days to 14 days, from 2.5 days to 10 days,from 2 days to 15 days, from 5 days to 15 days, from 10 days to 20 days,and so on.

As noted above, in some embodiments of any of the methods describedherein, the therapeutically effective amount of the C5 antagonistadministered to the human having a reduced C5 concentration is less thanthe therapeutically effective amount of the C5 antagonist requiredwithout the reduction in C5 concentration. The terms “therapeuticallyeffective amount” or “therapeutically effective dose,” or similar termsused herein are intended to mean an amount of an agent (e.g., atherapeutic agent such as a C5 antagonist) that will elicit the desiredbiological or medical response (e.g., an improvement in one or moresymptoms of a complement-associated disorder and/or inhibition ofcomplement activity).

In some embodiments of any of the methods described herein, thefrequency of administration of the C5 antagonist to the human having areduced C5 concentration is less often than the frequency ofadministration of the C5 antagonist to the human required without thereduction in C5 concentration. For example, in embodiments in which theC5 antagonist is an anti-C5 antibody, the frequency of administration ofthe C5 antagonist to the human having a reduced C5 concentration is nomore frequently than once monthly. In another example, in embodiments inwhich the C5 antagonist is an anti-C5 antibody, the frequency ofadministration of the C5 antagonist to the human having a reduced C5concentration is no more frequently than once every two months.

In some embodiments of any of the methods described herein, thecomplement-associated disorder is selected from the group consisting ofparoxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic-uremicsyndrome (aHUS), shiga toxin E. coli-related hemolytic uremic syndrome(STEC-HUS), dense deposit disease (DDD), C3 nephropathy, myastheniagravis, neuromyelitis optica, cold agglutinin disease (CAD),antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV),asthma, age-related macular degeneration (AMD), transplant rejection,Goodpasture's syndrome, glomerulonephritis, vasculitis, rheumatoidarthritis, dermatitis, systemic lupus erythematosus (SLE),Guillain-Barre syndrome (GBS), dermatomyositis, psoriasis, Graves'disease, Hashimoto's thyroiditis, type I diabetes, pemphigus, autoimmunehemolytic anemia (AIHA), idiopathic thrombocytopenic purpura (ITP),lupus nephritis, ischemia-reperfusion injury, thromboticthrombocytopenic purpura (TTP), Pauci-immune vasculitis, epidermolysisbullosa, multiple sclerosis, spontaneous fetal loss, recurrent fetalloss, traumatic brain injury, injury resulting from myocardialinfarction, cardiopulmonary bypass and hemodialysis, and hemolysis,elevated liver enzymes, and low platelets (HELLP) syndrome.

“Polypeptide,” “peptide,” and “protein” are used interchangeably andmean any peptide-linked chain of amino acids, regardless of length orpost-translational modification. As noted below, the polypeptidesdescribed herein can be, e.g., wild-type proteins, functional fragmentsof the wild-type proteins, or variants of the wild-type proteins orfragments. Variants, in accordance with the disclosure, can containamino acid substitutions, deletions, or insertions. The substitutionscan be conservative or non-conservative. Conservative substitutionstypically include substitutions within the following groups: glycine andalanine; valine, isoleucine, and leucine; aspartic acid and glutamicacid; asparagine, glutamine, serine and threonine; lysine, histidine andarginine; and phenylalanine and tyrosine.

As used herein, percent (%) amino acid sequence identity is defined asthe percentage of amino acids in a candidate sequence that are identicalto the amino acids in a reference sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity. Alignment for purposes of determining percentsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST software. Appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full-length of the sequences being compared can be determinedby known methods.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the presently disclosed methods and compositions.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Other features and advantages of the present disclosure, e.g., methodsfor increasing the half-life of a therapeutic agent (e.g., a C5antagonist), will be apparent from the following description, theexamples, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an exemplary nucleotide sequence for human complementcomponent C5 (SEQ ID NO:1).

FIG. 2 depicts an exemplary amino acid sequence for human complementcomponent pro-C5 (SEQ ID NO:2).

DETAILED DESCRIPTION

The disclosure features compositions (e.g., compounds and therapeuticagents such as C5 antagonists) and methods for prolonging the half-lifeof a therapeutic agent in the serum of a human. For example, thedisclosure provides methods for prolonging the serum half-life of a C5antagonist (e.g., an anti-C5 antibody) in a human by reducing theconcentration of C5 in the serum of the human. While in no way meant tobe limiting, exemplary compositions, conjugates, pharmaceuticalcompositions and formulations, methods for generating such compositions,as well as methods for using the compositions are set forth below.

Compounds that Reduce Serum Concentration of Complement Component C5

As used herein, “a compound that reduces the concentration of complementcomponent C5 in serum” is any agent that inhibits: (i) the expression ofa complement component C5 protein; (ii) the proper intracellulartrafficking, the post-translational modification, or secretion by acell, of a complement component C5 protein; or (iii) the stability of aC5 protein in the serum of a subject, which ultimately has the impact ofreducing the concentration of C5 in the serum of a subject (e.g., ahuman). Inhibition of complement component C5 protein expressionincludes: inhibition of transcription of a gene encoding a human C5protein; increased degradation of an mRNA encoding a human C5 protein;and/or inhibition of translation of an mRNA encoding a human C5 protein.Accordingly, a compound can reduce serum C5 concentration through, amongother things, increased degradation of a human C5 protein; inhibition ofproper processing of a pre-pro human C5 protein; or inhibition of propertrafficking or secretion by a cell of a human C5 protein. In someembodiments, the compound does not include an antibody that binds to C5.The compound can be, e.g., a small molecule, a nucleic acid (e.g., ansiRNA or an antisense oligonucleotide), a protein, or an aptamer.

In some embodiments, the compound is one that inhibits expression of C5protein. Nucleic acid inhibitors, e.g., can be used to decreaseexpression of an endogenous gene encoding human complement component C5.The nucleic acid antagonist can be, e.g., an siRNA, a dsRNA, a ribozyme,a triple-helix former, an aptamer, or an antisense nucleic acid. siRNAsare small double stranded RNAs (dsRNAs) that optionally includeoverhangs. For example, the duplex region of an siRNA can be about 18 to25 nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24nucleotides in length. The siRNA sequences can be, in some embodiments,exactly complementary to the target mRNA. dsRNAs and siRNAs inparticular can be used to silence gene expression in mammalian cells(e.g., human cells). One of skill in the art would understand how todesign such inhibitory nucleic acids in view of the human C5 codingsequence (e.g., SEQ ID NO:1). See, e.g., Clemens et al. (2000) Proc.Natl. Acad. Sci. USA 97:6499-6503; Billy et al. (2001) Proc. Natl. Acad.Sci. USA 98:14428-14433; Elbashir et al. (2001) Nature 411:494-8; Yanget al. (2002) Proc. Natl. Acad. Sci. USA 99:9942-9947, and U.S. patentapplication publication nos. 20030166282, 20030143204, 20040038278, and20030224432. Anti-sense agents can include, for example, from about 8 toabout 80 nucleobases (i.e. from about 8 to about 80 nucleotides), e.g.,about 8 to about 50 nucleobases, or about 12 to about 30 nucleobases.Anti-sense compounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression. Anti-sense compounds can include a stretchof at least eight consecutive nucleobases that are complementary to asequence in the target gene. An oligonucleotide need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target interferes with the normalfunction of the target molecule to cause a loss of utility, and there isa sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment or, in the case of invitro assays, under conditions in which the assays are conducted.Hybridization of antisense oligonucleotides with mRNA (e.g., an mRNAencoding a human C5 protein) can interfere with one or more of thenormal functions of mRNA. The functions of mRNA to be interfered withinclude all key functions such as, for example, translocation of the RNAto the site of protein translation, translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and catalyticactivity which may be engaged in by the RNA. Binding of specificprotein(s) to the RNA may also be interfered with by antisenseoligonucleotide hybridization to the RNA. Exemplary antisense compoundsinclude DNA or RNA sequences that specifically hybridize to the targetnucleic acid, e.g., the mRNA encoding a human complement component C5protein. The complementary region can extend for between about 8 toabout 80 nucleobases. The compounds can include one or more modifiednucleobases.

Modified nucleobases may include, e.g., 5-substituted pyrimidines suchas 5-iodouracil, 5-iodocytosine, and C₅-propynyl pyrimidines such asC₅-propynylcytosine and C₅-propynyluracil. Other suitable modifiednucleobases include, e.g., 7-substituted-8-aza-7-deazapurines and7-substituted-7-deazapurines such as, for example,7-iodo-7-deazapurines, 7-cyano-7-deazapurines, and7-aminocarbonyl-7-deazapurines. Examples of these include6-amino-7-iodo-7-deazapurines, 6-amino-7-cyano-7-deazapurines,6-amino-7-aminocarbonyl-7-deazapurines,2-amino-6-hydroxy-7-iodo-7-deazapurines,2-amino-6-hydroxy-7-cyano-7-deazapurines, and2-amino-6-hydroxy-7-aminocarbonyl-7-deazapurines. See, e.g., U.S. Pat.Nos. 4,987,071; 5,116,742; and 5,093,246; “Antisense RNA and DNA,” D. A.Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1988); Haselhoff and Gerlach (1988) Nature 334:585-59; Helene (1991)Anticancer Drug D 6:569-84; Helene (1992) Ann. NY. Acad. Sci. 660:27-36;and Maher (1992) Bioassays 14:807-15.

Methods for determining whether a compound has inhibited the expressionof an antigen (e.g., C5 protein) are well known in the art. Such methodsfor detecting protein expression include, without limitation: Westernblot, dot blot, enzyme-linked immunosorbent assay (ELISA), “sandwich”immunoassays, immunoprecipitation assays, AlphaScreen® or AlphaLISA®assays, or mass spectrometry based methods.

The term “immunoassay” encompasses techniques including, withoutlimitation, flow cytometry, FACS, enzyme immunoassays (EIA), such asenzyme multiplied immunoassay technique (EMIT), enzyme-linkedimmunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA) andmicroparticle enzyme immunoassay (MEIA), furthermore capillaryelectrophoresis immunoassays (CEIA), radio-immunoassays (RIA),immunoradiometric assays (IRMA), fluorescence polarization immunoassays(FPIA) and chemiluminescence assays (CL). If desired, such immunoassayscan be automated. Immunoassays can also be used in conjunction withlaser induced fluorescence. Liposome immunoassays, such asflow-injection liposome immunoassays and liposome immunosensors, arealso suitable for use in the present invention. In addition,nephelometry assays, in which, for example, the formation ofprotein/antibody complexes results in increased light scatter that isconverted to a peak rate signal as a function of the markerconcentration, are suitable for use in the methods of the presentinvention. In a preferred embodiment of the present invention, theincubation products are detected by ELISA, RIA, fluoro immunoassay (FIA)or soluble particle immune assay (SPIA).

Generally the amount of C5 in a serum sample from a patient taken beforeadministration of the compound is compared to the amount of C5 in aserum sample from the same patient following administration of thecompound (e.g., 6 hours, 12 hours, 1 day, 36 hours, 2 days, 3 days, 1week or 2 weeks after administration of the compound) to the patient. Areduced amount of C5 in the serum sample obtained after administrationof the compound, as compared to the amount of C5 in an equivalent serumsample obtained from the patient prior to administration of thecompound, indicates that the compound is one that reduces theconcentration of C5 in the serum of the patient.

C5 Antagonists

A C5 antagonist can be, e.g., a small molecule, a polypeptide, apolypeptide analog, a nucleic acid, or a nucleic acid analog. “Smallmolecule” as used herein, is meant to refer to an agent, whichpreferably has a molecular weight of less than about 6 kDa and mostpreferably less than about 2.5 kDa. Many pharmaceutical companies haveextensive libraries of chemical and/or biological mixtures comprisingarrays of small molecules, often fungal, bacterial, or algal extracts,which can be screened with any of the assays of the application. Thisapplication contemplates using, among other things, small chemicallibraries, peptide libraries, or collections of natural products. Tan etal. described a library with over two million synthetic compounds thatis compatible with miniaturized cell-based assays (J Am Chem Soc (1998)120:8565-8566). It is within the scope of this application that such alibrary may be used to screen for inhibitors of human complementcomponent C5. There are numerous commercially available compoundlibraries, such as the Chembridge DIVERSet. Libraries are also availablefrom academic investigators, such as the Diversity set from the NCIdevelopmental therapeutics program. Rational drug design may also beemployed. For example, rational drug design can employ the use ofcrystal or solution structural information on the human complementcomponent C5 protein. See, e.g., the structures described in Hagemann etal. (2008) J Biol Chem 283(12):7763-75 and Zuiderweg et al. (1989)Biochemistry 28(1):172-85. See also, Fredslund et al. (2008) Nat.Immunol. 9:753-760 and Laursen et al. (2011) EMBO J. 30:606-616.Rational drug design can also be achieved based on known compounds,e.g., a known inhibitor of C5 (e.g., an antibody, or antigen-bindingfragment thereof, that binds to a human complement component C5protein). The amino acid sequence of human C5 is known (see, Haviland etal. (1991) J. Immunol. 146:362-368) and is shown in FIG. 2 as SEQ IDNO:2. Human C5 is synthesized as a pro-05 molecule including an 18 aminoacid signal peptide plus a 1658 amino acid protein that gets processedby cleavage between amino acids 655-656 and 659-660. Once fullyprocessed, the processing results in an 18 amino acid peptidecorresponding to the signal peptide, a 655 amino acid peptide (aminoacids 1-655) that is referred to as the β-chain, a 4 amino acid peptidecorresponding to amino acids 656-659 which is lost, and a 999 amino acidpeptide corresponding to amino acids 660-1658, this being referred to asthe α-chain. The α-chain and β-chain become linked to each other viadisulfide bonds. This final structure of the α- and β-chains is thecomplete C5 molecule. This C5 can be cleaved by a C5 convertase by acleavage between amino acids 733-734 thereby cleaving off a 74-aminoacid fragment from the α-chain (corresponding to amino acids 660-733).This 74 amino acid fragment is C5a.

Peptidomimetics can be compounds in which at least a portion of asubject polypeptide is modified, and the three dimensional structure ofthe peptidomimetic remains substantially the same as that of the subjectpolypeptide. Peptidomimetics may be analogues of a subject polypeptideof the disclosure that are, themselves, polypeptides containing one ormore substitutions or other modifications within the subject polypeptidesequence. Alternatively, at least a portion of the subject polypeptidesequence may be replaced with a non-peptide structure, such that thethree-dimensional structure of the subject polypeptide is substantiallyretained. In other words, one, two or three amino acid residues withinthe subject polypeptide sequence may be replaced by a non-peptidestructure. In addition, other peptide portions of the subjectpolypeptide may, but need not, be replaced with a non-peptide structure.Peptidomimetics (both peptide and non-peptidyl analogues) may haveimproved properties (e.g., decreased proteolysis, increased retention orincreased bioavailability). Peptidomimetics generally have improved oralavailability, which makes them especially suited to treatment ofdisorders in a human or animal. It should be noted that peptidomimeticsmay or may not have similar two-dimensional chemical structures, butshare common three-dimensional structural features and geometry. Eachpeptidomimetic may further have one or more unique additional bindingelements.

Aptamers are short oligonucleotide sequences that can be used torecognize and specifically bind almost any molecule, including cellsurface proteins. The systematic evolution of ligands by exponentialenrichment (SELEX) process is powerful and can be used to readilyidentify such aptamers. Aptamers can be made for a wide range ofproteins of importance for therapy and diagnostics, such as growthfactors and cell surface antigens. These oligonucleotides bind theirtargets with similar affinities and specificities as antibodies do (see,e.g., Ulrich (2006) Handb Exp Pharmacol. 173:305-326).

In some embodiments, the inhibitor of human C5 is an antibody, orantigen-binding fragment thereof, which binds to a human complementcomponent C5 protein. (Hereinafter, the antibody may sometimes bereferred to as an “anti-C5 antibody.”)

In some embodiments, the C5 antagonist comprises, and/or is, eculizumab(Soliris®; Alexion Pharmaceuticals, Inc., Cheshire, Conn.). See, e.g.,Kaplan (2002) Curr Opin Investig Drugs 3(7):1017-23; Hill (2005) ClinAdv Hematol Oncol 3(11):849-50; and Rother et al. (2007) NatureBiotechnology 25(11):1256-1488. In some embodiments, the C5 antagonistcomprises, and/or is, pexelizumab (Alexion Pharmaceuticals, Inc.,Cheshire, Conn.). See, e.g., Whiss (2002) Curr Opin Investig Drugs3(6):870-7; Patel et al. (2005) Drugs Today (Barc) 41(3):165-70; andThomas et al. (1996) Mol Immunol. 33(17-18):1389-401. In someembodiments, the anti-C5 antibody or antigen-binding fragment thereofcan have the same or greater affinity for C5 as does eculizumab orpexelizumab.

In some embodiments, the anti-C5 antibody or antigen-binding fragmentthereof can bind to an epitope in the alpha chain of the humancomplement component C5 protein. Antibodies that bind to the alpha chainof C5 are described in, for example, International patent applicationpublication no. WO 2010/136311 and U.S. Pat. No. 6,355,245. In someembodiments, the anti-C5 antibody can bind to an epitope in the betachain of the human complement component C5 protein. Antibodies that bindto the C5 beta chain are described in, e.g., Moongkarndi et al. (1982)Immunobiol 162:397; Moongkarndi et al. (1983) Immunobiol 165:323; andMollnes et al. (1988) Scand J Immunol 28:307-312. See also Internationalpatent application publication no. WO 2010/136311.

In some embodiments, the C5 antagonist can be a non-antibody, scaffoldprotein. These proteins are, generally, obtained through combinatorialchemistry-based adaptation of pre-existing antigen-binding proteins. Forexample, the binding site of human transferrin for human transferrinreceptor can be modified using combinatorial chemistry to create adiverse library of transferrin variants, some of which have acquiredaffinity for different antigens. Ali et al. (1999) J Biol Chem274:24066-24073. The portion of human transferrin not involved withbinding the receptor remains unchanged and serves as a scaffold, likeframework regions of antibodies, to present the variant binding sites.The libraries are then screened, as an antibody library is, against atarget antigen of interest to identify those variants having optimalselectivity and affinity for the target antigen. Non-antibody scaffoldproteins, while similar in function to antibodies, are touted as havinga number of advantages as compared to antibodies, which advantagesinclude, among other things, enhanced solubility and tissue penetration,less costly manufacture, and ease of conjugation to other molecules ofinterest. Hey et al. (2005) TRENDS Biotechnol 23(10):514-522.

One of skill in the art would appreciate that the scaffold portion ofthe non-antibody scaffold protein can include, e.g., all or part of: theZ domain of S. aureus protein A, human transferrin, human tenthfibronectin type III domain, kunitz domain of a human trypsin inhibitor,human CTLA-4, an ankyrin repeat protein, a human lipocalin, humancrystallin, human ubiquitin, or a trypsin inhibitor from E. elaterium.Id.

Other exemplary C5 antagonists include the anti-C5 minibody MB12/22(Mubodina®; Adienne Pharma & Biotech, Bergamo, Italy) and a variant formof the minibody fused with RGD peptide, MB12/22-RGD (Ergidina®; AdiennePharma & Biotech, Bergamo, Italy). MB12/22 and MB12/22-RGD are derivedfrom an anti-C5 scFv, Ts-a12/22, which is described in Internationalpatent application publication no. WO 2004/007553. MB-12/22 andMB-12/22-RGD recognize an epitope comprising the C5 convertase cleavagesite located between amino acids 733 and 734 of the C5 polypeptide (SEQID NO:2). Other anti-C5 antibodies that variously recognize epitopes oneither the alpha chain or the beta chain of the C5 molecule and inhibitcomplement mediated hemolytic activity, are described in Internationalpatent application publication no. WO 2010/015608. C5 binding aptamers,ARC187 and ARC1905 (commercially available from Archemix/OphthotechCorp., Princeton, N.J.), are described in U.S. patent applicationpublication no. 20070048248. OmCI, a protein excreted by the soft tickOrnithodoros moubata, is a naturally occurring inhibitor of C5 activity.A recombinant variant of OmCI, rev576, has also been described (Hepburnet al. (2007) J Biol Chem 282:8292-8299 and Soltys et al. (2009) AnnNeurol 65:67-75). Another naturally occurring inhibitor of C5 activityis the Staphylococcus aureus secreted protein SSL7 (Laursen et al.(2010) Proc. Natl. Acad. Sci. 107:3681-3686).

Methods for determining whether an agent is a C5 antagonist are wellknown in the art. The C5 antagonists described herein have activity inblocking the generation or activity of the C5a and/or C5b activefragments of a complement component C5 protein (e.g., a human C5protein). Through this blocking effect, the C5 antagonists inhibit,e.g., the proinflammatory effects of C5a and the generation of the C5b-9membrane attack complex (MAC) at the surface of a cell.

Inhibition of human complement component C5 can also reduce thecell-lysing ability of complement in a subject's body fluids. Suchreductions of the cell-lysing ability of complement present in the bodyfluid(s) can be measured by methods well known in the art such as, forexample, by a conventional hemolytic assay such as the hemolysis assaydescribed by Kabat and Mayer (eds.), “Experimental Immunochemistry,2^(nd) Edition,” 135-240, Springfield, Ill., CC Thomas (1961), pages135-139, or a conventional variation of that assay such as the chickenerythrocyte hemolysis method as described in, e.g., Hillmen et al.(2004) N Engl J Med 350(6):552. Methods for determining whether an agentinhibits the cleavage of human C5 into forms C5a and C5b are known inthe art and described in, e.g., Moongkarndi et al. (1982) Immunobiol.162:397; Moongkarndi et al. (1983) Immunobiol. 165:323; Isenman et al.(1980) J Immunol. 124(1):326-31; Thomas et al. (1996) Mol. Immunol.33(17-18):1389-401; and Evans et al. (1995) Mol. Immunol.32(16):1183-95. For example, the concentration and/or physiologicactivity of C5a and C5b in a body fluid can be measured by methods wellknown in the art. Methods for measuring C5a concentration or activityinclude, e.g., chemotaxis assays, RIAs, or ELISAs (see, e.g., Ward andZvaifler (1971) J Clin Invest. 50(3):606-16 and Wurzner et al. (1991)Complement Inflamm. 8:328-340). For C5b, hemolytic assays or assays forsoluble C5b-9 as discussed herein can be used. Other assays known in theart can also be used. Using assays of these or other suitable types,agents capable of inhibiting human complement component C5 can bescreened.

Immunological techniques such as, but not limited to, ELISA can be usedto measure the protein concentration of C5 and/or its split products todetermine the ability of a test compound to inhibit conversion of C5into biologically active products. In some embodiments, C5a generationis measured. In some embodiments, C5b-9 neoepitope-specific antibodiesare used to detect the formation of terminal complement.

Hemolytic assays can be used to determine the inhibitory activity of aputative C5 antagonist on complement activation. In order to determinethe effect of a C5 antagonist on classical complement pathway-mediatedhemolysis in a serum test solution in vitro, for example, sheeperythrocytes coated with hemolysin or chicken erythrocytes sensitizedwith anti-chicken erythrocyte antibody are used as target cells. Thepercentage of lysis is normalized by considering 100% lysis equal to thelysis occurring in the absence of the inhibitor. In some embodiments,the classical complement pathway is activated by a human IgM antibody,for example, as utilized in the Wieslab® Classical Pathway ComplementKit (Wieslab® COMPL CP310, Euro-Diagnostica, Sweden). Briefly, the testserum is incubated with a test compound in the presence of a human IgMantibody. The amount of C5b-9 that is generated is measured bycontacting the mixture with an enzyme conjugated anti-05b-9 antibody anda fluorogenic substrate and measuring the absorbance at the appropriatewavelength. As a control, the test serum is incubated in the absence ofthe putative C5 antagonist. In some embodiments, the test serum is aC5-deficient serum reconstituted with a C5 polypeptide.

To determine the effect of putative C5 antagonist on alternativepathway-mediated hemolysis, unsensitized rabbit or guinea pigerythrocytes are used as the target cells. In some embodiments, theserum test solution is a C5-deficient serum reconstituted with a C5polypeptide. The percentage of lysis is normalized by considering 100%lysis equal to the lysis occurring in the absence of the inhibitor. Insome embodiments, the alternative complement pathway is activated bylipopolysaccharide molecules, for example, as utilized in the Wieslab®Alternative Pathway Complement Kit (Wieslab® COMPL AP330,Euro-Diagnostica, Sweden). Briefly, the test serum is incubated with atest compound in the presence of lipopolysaccharide. The amount of C5b-9that is generated is measured by contacting the mixture with an enzymeconjugated anti-05b-9 antibody and a fluorogenic substrate and measuringthe fluorescence at the appropriate wavelength. As a control, the testserum is incubated in the absence of the test compound.

In some embodiments, C5 activity, or inhibition thereof, is quantifiedusing a CH50eq assay. The CH50eq assay is a method for measuring thetotal classical complement activity in serum. This test is a lyticassay, which uses antibody-sensitized erythrocytes as the activator ofthe classical complement pathway and various dilutions of the test serumto determine the amount required to give 50% lysis (CH50). The percenthemolysis can be determined, for example, using a spectrophotometer. TheCH50eq assay provides an indirect measure of terminal complement complex(TCC) formation, since the TCC themselves are directly responsible forthe hemolysis that is measured.

The assay is well known and commonly practiced by those of skill in theart. Briefly, to activate the classical complement pathway, undilutedserum samples (e.g., reconstituted human serum samples) are added tomicroassay wells containing the antibody-sensitized erythrocytes tothereby generate TCC. Next, the activated sera are diluted in microassaywells, which are coated with a capture reagent (e.g., an antibody thatbinds to one or more components of the TCC). The TCC present in theactivated samples bind to the monoclonal antibodies coating the surfaceof the microassay wells. The wells are washed and to each well is addeda detection reagent that is detectably labeled and recognizes the boundTCC. The detectable label can be, e.g., a fluorescent label or anenzymatic label. The assay results are expressed in CH50 unitequivalents per milliliter (CH50 U Eq/mL).

Methods for determining the half-life of a therapeutic agent (e.g., a C5antagonist such as an anti-C5 antibody), as well as changes in half-life(e.g., increases in half-life), are well known in the art. See, e.g.,International patent application publication no. WO 2010/151526;International patent application publication no. WO 98/23289;International patent application publication no. WO 97/34631; U.S. Pat.No. 6,277,375; International patent application publication nos. WO93/15199, WO 93/15200, and WO 01/77137; European Patent No. EP 413 622;and U.S. patent application publication no. 20110111406. See also Hintonet al. (2006) J Immunol 176:346-356.

Methods for Treatment

The methods described herein include administering to a subject (e.g., ahuman) a compound that reduces the concentration of an antigen to whicha therapeutic agent binds (e.g., human complement component C5) and, inthe context of the reduced concentration of the antigen (e.g., C5),administering to the human the therapeutic agent (e.g., a C5 antagonistsuch as an anti-C5 antibody).

The compounds and agents (e.g., C5 antagonists) described herein can beadministered to a subject, e.g., a human subject, using a variety ofmethods that depend, in part, on the route of administration. The routecan be, e.g., intravenous injection or infusion (IV), subcutaneousinjection (SC), intraperitoneal (IP) injection, or intramuscularinjection (IM).

Administration can be achieved by, e.g., local infusion, injection, orby means of an implant. The implant can be of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. The implant can be configured for sustained or periodicrelease of the composition to the subject. See, e.g., U.S. PatentApplication Publication No. 20080241223; U.S. Pat. Nos. 5,501,856;4,863,457; and 3,710,795; EP488401; and EP430539, the disclosures ofeach of which are incorporated herein by reference in their entirety. Acomposition described herein (e.g., a compound and/or a C5 antagonist)can be delivered to the subject by way of an implantable device basedon, e.g., diffusive, erodible, or convective systems, e.g., osmoticpumps, biodegradable implants, electrodiffusion systems, electroosmosissystems, vapor pressure pumps, electrolytic pumps, effervescent pumps,piezoelectric pumps, erosion-based systems, or electromechanicalsystems.

In some embodiments, a composition described herein is therapeuticallydelivered to a subject by way of local administration. As used herein,“local administration” or “local delivery,” refers to delivery that doesnot rely upon transport of the composition or agent to its intendedtarget tissue or site via the vascular system. For example, thecomposition may be delivered by injection or implantation of thecomposition or agent or by injection or implantation of a devicecontaining the composition or agent. Following local administration inthe vicinity of a target tissue or site, a C5 antagonist, e.g., maydiffuse to the intended target tissue or site.

In some embodiments, a composition described herein can be locallyadministered to a joint (e.g., an articulated joint). For example, inembodiments where the disorder is arthritis, a therapeuticallyappropriate composition can be administered directly to a joint (e.g.,into a joint space) or in the vicinity of a joint. Examples ofintraarticular joints to which a composition described herein can belocally administered include, e.g., the hip, knee, elbow, wrist,sternoclavicular, temperomandibular, carpal, tarsal, ankle, and anyother joint subject to arthritic conditions. A composition describedherein can also be administered to bursa such as, e.g., acromial,bicipitoradial, cubitoradial, deltoid, infrapatellar, ischial, and anyother bursa known in the art of medicine.

In some embodiments, a composition described herein can be locallyadministered to the eye. As used herein, the term “eye” refers to anyand all anatomical tissues and structures associated with an eye. Theeye has a wall composed of three distinct layers: the outer sclera, themiddle choroid layer, and the inner retina. The chamber behind the lensis filled with a gelatinous fluid referred to as the vitreous humor. Atthe back of the eye is the retina, which detects light. The cornea is anoptically transparent tissue, which conveys images to the back of theeye. The cornea includes one pathway for the permeation of drugs intothe eye. Other anatomical tissue structures associated with the eyeinclude the lacrimal drainage system, which includes a secretory system,a distributive system and an excretory system. The secretory systemcomprises secretors that are stimulated by blinking and temperaturechange due to tear evaporation and reflex secretors that have anefferent parasympathetic nerve supply and secrete tears in response tophysical or emotional stimulation. The distributive system includes theeyelids and the tear meniscus around the lid edges of an open eye, whichspread tears over the ocular surface by blinking, thus reducing dryareas from developing.

In some embodiments, a composition (e.g., one or both of the compoundand a C5 antagonist) described herein is administered to the posteriorchamber of the eye. In some embodiments, a composition described hereinis administered intravitreally. In some embodiments, a compositiondescribed herein is administered trans-sclerally.

In some embodiments, e.g., in embodiments for treatment or prevention ofa disorder such as COPD or asthma, a composition (e.g., a C5 antagonist)described herein can be administered to a subject by way of the lung.Pulmonary drug delivery may be achieved by inhalation, andadministration by inhalation herein may be oral and/or nasal. Examplesof pharmaceutical devices for pulmonary delivery include metered doseinhalers, dry powder inhalers (DPIs), and nebulizers. For example, acomposition described herein can be administered to the lungs of asubject by way of a dry powder inhaler. These inhalers arepropellant-free devices that deliver dispersible and stable dry powderformulations to the lungs. Dry powder inhalers are well known in the artof medicine and include, without limitation: the TurboHaler®(AstraZeneca; London, England); the AIR® inhaler (Alkermes®; Cambridge,Mass.); Rotahaler® (GlaxoSmithKline; London, England); and Eclipse™(Sanofi-Aventis; Paris, France). See also, e.g., PCT Publication Nos. WO04/026380, WO 04/024156, and WO 01/78693. DPI devices have been used forpulmonary administration of polypeptides such as insulin and growthhormone. In some embodiments, a composition described herein can beintrapulmonarily administered by way of a metered dose inhaler. Theseinhalers rely on a propellant to deliver a discrete dose of a compoundto the lungs.

In some embodiments, a composition (e.g., a C5 antagonist) describedherein can be administered to the lungs of a subject by way of anebulizer. Nebulizers use compressed air to deliver a compound as aliquefied aerosol or mist. A nebulizer can be, e.g., a jet nebulizer(e.g., air or liquid-jet nebulizers) or an ultrasonic nebulizer.Additional devices and intrapulmonary administration methods are setforth in, e.g., U.S. Patent Application Publication Nos. 20050271660 and20090110679, the disclosures of each of which are incorporated herein byreference in their entirety.

In some embodiments, the compositions provided herein are present inunit dosage form, which can be particularly suitable forself-administration. For example, in some embodiments the compound thatreduces the serum concentration of an antigen (e.g., human C5) can beformulated for self-administration (e.g., self-subcutaneousadministration). A formulated product of the present disclosure can beincluded within a container, typically, for example, a vial, cartridge,prefilled syringe or disposable pen. A doser such as the doser devicedescribed in U.S. Pat. No. 6,302,855 may also be used, for example, withan injection system of the present disclosure.

An injection system of the present disclosure may employ a delivery penas described in U.S. Pat. No. 5,308,341. Pen devices, most commonly usedfor self-delivery of insulin to patients with diabetes, are well knownin the art. Such devices can comprise at least one injection needle(e.g., a 31 gauge needle of about 5 to 8 mm in length), are typicallypre-filled with one or more therapeutic unit doses of a therapeuticsolution, and are useful for rapidly delivering the solution to asubject with as little pain as possible.

One medication delivery pen includes a vial holder into which a vial ofinsulin or other medication may be received. The vial holder is anelongate generally tubular structure with proximal and distal ends. Thedistal end of the vial holder includes mounting means for engaging adouble-ended needle cannula. The proximal end also includes mountingmeans for engaging a pen body which includes a driver and dose settingapparatus. A disposable medication (e.g., a high concentration solutionof a composition described herein) containing vial for use with theprior art vial holder includes a distal end having a pierceableelastomeric septum that can be pierced by one end of a double-endedneedle cannula. The proximal end of this vial includes a stopperslidably disposed in fluid tight engagement with the cylindrical wall ofthe vial. This medication delivery pen is used by inserting the vial ofmedication into the vial holder. A pen body then is connected to theproximal end of the vial holder. The pen body includes a dose settingapparatus for designating a dose of medication to be delivered by thepen and a driving apparatus for urging the stopper of the vial distallyfor a distance corresponding to the selected dose. The user of the penmounts a double-ended needle cannula to the distal end of the vialholder such that the proximal point of the needle cannula pierces theseptum on the vial. The patient then selects a dose and operates the pento urge the stopper distally to deliver the selected dose. The doseselecting apparatus returns to zero upon injection of the selected dose.The patient then removes and discards the needle cannula, and keeps themedication delivery pen in a convenient location for the next requiredmedication administration. The medication in the vial will becomeexhausted after several such administrations of medication. The patientthen separates the vial holder from the pen body. The empty vial maythen be removed and discarded. A new vial can be inserted into the vialholder, and the vial holder and pen body can be reassembled and used asexplained above. Accordingly, a medication delivery pen generally has adrive mechanism for accurate dosing and ease of use.

A dosage mechanism such as a rotatable knob allows the user toaccurately adjust the amount of medication that will be injected by thepen from a prepackaged vial of medication. To inject the dose ofmedication, the user inserts the needle under the skin and depresses theknob once as far as it will depress. The pen may be an entirelymechanical device or it may be combined with electronic circuitry toaccurately set and/or indicate the dosage of medication that is injectedinto the user. See, e.g., U.S. Pat. No. 6,192,891.

In some embodiments, the needle of the pen device is disposable and thekits include one or more disposable replacement needles. Pen devicessuitable for delivery of any one of the presently featured compositionsare also described in, e.g., U.S. Pat. Nos. 6,277,099; 6,200,296; and6,146,361, the disclosures of each of which are incorporated herein byreference in their entirety. A microneedle-based pen device is describedin, e.g., U.S. Pat. No. 7,556,615, the disclosure of which isincorporated herein by reference in its entirety. See also the PrecisionPen Injector (PPI) device, Molly™, manufactured by Scandinavian HealthLtd.

The present disclosure also presents controlled-release orextended-release formulations suitable for chronic and/orself-administration of a medication such as a composition (e.g., acompound or a C5 antagonist) described herein. The various formulationscan be administered to a patient in need of treatment with themedication as a bolus or by continuous infusion over a period of time.

In some embodiments, a high concentration composition (e.g., a highconcentration compound or C5 antagonist) described herein is formulatedfor sustained-release, extended-release, timed-release,controlled-release, or continuous-release administration. In someembodiments, depot formulations are used to administer the compositionto the subject in need thereof. In this method, the composition isformulated with one or more carriers providing a gradual release ofactive agent over a period of a number of hours or days. Suchformulations are often based upon a degrading matrix which graduallydisperses in the body to release the active agent.

As noted above, in some embodiments the compound and the therapeuticagent (e.g., the C5 antagonist) can be administered to a human bydifferent routes. For example, the compound such as an siRNA thattargets C5 can be administered subcutaneously (e.g., once weekly) andthe C5 antagonist (e.g., an anti-C5 antibody such as eculizumab) can beadministered intravenously (e.g., once monthly or once every twomonths).

A suitable dose of a given composition described herein, which dose iscapable of treating or preventing a disorder in a subject, can depend ona variety of factors including, e.g., the age, sex, and weight of asubject to be treated and the particular inhibitor compound used. Otherfactors can include, e.g., other medical disorders concurrently orpreviously affecting the subject, the general health of the subject, thegenetic disposition of the subject, diet, time of administration, rateof excretion, drug combination, and any other additional therapeuticsthat are administered to the subject. It should also be understood thata specific dosage and treatment regimen for any particular subject willalso depend upon the judgment of the treating medical practitioner(e.g., doctor or nurse).

A composition described herein can be administered as a fixed dose, orin a milligram per kilogram (mg/kg) dose. In some embodiments, the dosecan also be chosen to reduce or avoid production of antibodies or otherhost immune responses against a compound or a therapeutic agent (such asa C5 antagonist). While in no way intended to be limiting, exemplarydosages of an antibody, such as a composition described herein include,e.g., 1-1000 mg/kg, 1-100 mg/kg, 0.5-50 mg/kg, 0.1-100 mg/kg, 0.5-25mg/kg, 1-20 mg/kg, and 1-10 mg/kg. Exemplary dosages of a compositiondescribed herein include, without limitation, 0.1 mg/kg, 0.5 mg/kg, 1.0mg/kg, 2.0 mg/kg, 4 mg/kg, 8 mg/kg, or 20 mg/kg.

A pharmaceutical composition can include a therapeutically effectiveamount of a composition described herein. Such effective amounts can bereadily determined by one of ordinary skill in the art based, in part,on the effect of the administered composition, or the combinatorialeffect of the composition and one or more additional active agents, ifmore than one agent is used. A therapeutically effective amount of acomposition described herein can also vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the composition (and one or more additional active agents) toelicit a desired response in the individual, e.g., amelioration of atleast one condition parameter, e.g., amelioration of at least onesymptom of the complement-mediated disorder. For example, atherapeutically effective amount of a composition described herein caninhibit (lessen the severity of or eliminate the occurrence of) and/orprevent a particular disorder, and/or any one of the symptoms of theparticular disorder known in the art or described herein. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the composition are outweighed by thetherapeutically beneficial effects.

Suitable human doses of any of the compositions described herein (e.g.,a compound or a C5 antagonist) can further be evaluated in, e.g., PhaseI dose escalation studies. See, e.g., van Gurp et al. (2008) Am JTransplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res13(2, part 1):523-531; and Hetherington et al. (2006) AntimicrobialAgents and Chemotherapy 50(10): 3499-3500. For example, suitable dosagesand/or frequency of dosages of a compound required to reduce serum C5concentration can be determined using such methods.

Toxicity and therapeutic efficacy of such compositions (e.g., thecompound or the C5 antagonist) can be determined by known pharmaceuticalprocedures in cell cultures or experimental animals (e.g., animal modelsof any of the complement-mediated disorders described herein). Theseprocedures can be used, e.g., for determining the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. Compositions described herein that exhibit a hightherapeutic index are preferred. While compositions that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue and tominimize potential damage to normal cells and, thereby, reduce sideeffects.

Data obtained from cell culture assays and animal studies can be used informulating a range of dosage for use in humans. The dosage of thecomposition described herein lies generally within a range ofcirculating concentrations of the compositions that include the ED₅₀with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For a composition described herein, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose can be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe antibody which achieves a half-maximal inhibition of symptoms) asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography. Insome embodiments, e.g., where local administration (e.g., to the eye ora joint) is desired, cell culture or animal modeling can be used todetermine a dose required to achieve a therapeutically effectiveconcentration within the local site.

In some embodiments, the methods can be performed in conjunction withother therapies for complement-associated disorders. For example, thecomposition can be administered to a subject at the same time, prior to,or after, plasmapheresis, IVIG therapy, or plasma exchange. See, e.g.,Appel et al. (2005) J Am Soc Nephrol 16:1392-1404. In some embodiments,the composition can be administered to a subject at the same time, priorto, or after, a kidney transplant.

A “subject,” as used herein, can be any mammal. For example, a subjectcan be a human, a non-human primate (e.g., orangutan, gorilla, macaque,baboon, or chimpanzee), a horse, a cow, a pig, a sheep, a goat, a dog, acat, a rabbit, a guinea pig, a gerbil, a hamster, a rat, or a mouse. Insome embodiments, the subject is an infant (e.g., a human infant).

As used herein, a subject “in need of prevention,” “in need oftreatment,” or “in need thereof,” refers to one, who by the judgment ofan appropriate medical practitioner (e.g., a doctor, a nurse, or a nursepractitioner in the case of humans; a veterinarian in the case ofnon-human mammals), would reasonably benefit from a given treatment.

The term “preventing” is art-recognized, and when used in relation to acondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive a composition described herein. Thus, prevention of acomplement-associated disorder such as asthma includes, for example,reducing the extent or frequency of coughing, wheezing, or chest pain ina population of patients receiving a prophylactic treatment relative toan untreated control population, and/or delaying the occurrence ofcoughing or wheezing in a treated population versus an untreated controlpopulation, e.g., by a statistically and/or clinically significantamount.

As described above, the compositions described herein (e.g., C5antagonists such an anti-C5 antibodies) can be used to treat a varietyof complement-associated disorders such as, but not limited to:rheumatoid arthritis (RA); lupus nephritis; ischemia-reperfusion injury;atypical hemolytic uremic syndrome (aHUS); typical or infectioushemolytic uremic syndrome (tHUS); dense deposit disease (DDD);paroxysmal nocturnal hemoglobinuria (PNH); multiple sclerosis (MS);macular degeneration (e.g., age-related macular degeneration (AMD));hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome;sepsis; dermatomyositis; diabetic retinopathy; thromboticthrombocytopenic purpura (TTP); spontaneous fetal loss; Pauci-immunevasculitis; epidermolysis bullosa; recurrent fetal loss; and traumaticbrain injury. See, e.g., Holers (2008) Immunological Reviews 223:300-316and Holers and Thurman (2004) Molecular Immunology 41:147-152. In someembodiments, the complement-mediated disorder is a complement-mediatedvascular disorder such as, but not limited to, a cardiovasculardisorder, myocarditis, a cerebrovascular disorder, a peripheral (e.g.,musculoskeletal) vascular disorder, a renovascular disorder, amesenteric/enteric vascular disorder, revascularization to transplantsand/or replants, vasculitis, Henoch-Schönlein purpura nephritis,systemic lupus erythematosus-associated vasculitis, vasculitisassociated with rheumatoid arthritis, immune complex vasculitis, organor tissue transplantation, Takayasu's disease, capillary leak syndrome,dilated cardiomyopathy, diabetic angiopathy, thoracic-abdominal aorticaneurysm, Kawasaki's disease (arteritis), venous gas embolus (VGE), andrestenosis following stent placement, rotational atherectomy, andpercutaneous transluminal coronary angioplasty (PTCA). (See, e.g., U.S.patent application publication no. 20070172483.) In some embodiments,the complement-associated disorder is myasthenia gravis, cold-agglutinindisease (CAD), paroxysmal cold hemoglobinuria (PCH), dermatomyositis,scleroderma, warm autoimmune hemolytic anemia, Graves' disease,Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus,autoimmune hemolytic anemia (AIHA), idiopathic thrombocytopenic purpura(ITP), Goodpasture's syndrome, antiphospholipid syndrome (APS), Degosdisease, and catastrophic APS (CAPS).

In some embodiments, a composition described herein can be used to treatan inflammatory disorder such as, but not limited to, RA (above),inflammatory bowel disease, sepsis (above), septic shock, acute lunginjury, disseminated intravascular coagulation (DIC), or Crohn'sdisease. In some embodiments, the second anti-inflammatory agent can beone selected from the group consisting of NSAIDs, corticosteroids,methotrexate, hydroxychloroquine, anti-TNF agents such as etanercept andinfliximab, a B cell depleting agent such as rituximab, an interleukin-1antagonist, or a T cell costimulatory blocking agent such as abatacept.

In some embodiments, the complement-associated disorder is acomplement-associated neurological disorder such as, but not limited to,amyotrophic lateral sclerosis (ALS), brain injury, Alzheimer's disease,and chronic inflammatory demyelinating neuropathy.

Complement-associated disorders also include complement-associatedpulmonary disorders such as, but not limited to, asthma, bronchitis, achronic obstructive pulmonary disease (COPD), an interstitial lungdisease, α-1 anti-trypsin deficiency, emphysema, bronchiectasis,bronchiolitis obliterans, alveolitis, sarcoidosis, pulmonary fibrosis,and collagen vascular disorders.

In some embodiments, a composition described herein is administered to asubject to treat, prevent, or ameliorate at least one symptom of acomplement-associated inflammatory response (e.g., thecomplement-associated inflammatory response aspect of acomplement-associated disorder) in a subject. For example, a compositioncan be used to treat, prevent, and/or ameliorate one or more symptomsassociated with a complement-associated inflammatory response such asgraft rejection/graft-versus-host disease (GVHD), reperfusion injuries(e.g., following cardiopulmonary bypass or a tissue transplant), andtissue damage following other forms of traumatic injury such as a burn(e.g., a severe burn), blunt trauma, spinal injury, or frostbite. See,e.g., Park et al. (1999) Anesth Analg 99(1):42-48; Tofukuji et al.(1998) J Thorac Cardiovasc Surg 116(6):1060-1068; Schmid et al. (1997)Shock 8(2):119-124; and Bless et al. (1999) Am J Physiol 276(1):L57-L63.

Monitoring a subject (e.g., a human patient) for an improvement in adisorder (e.g., sepsis, severe burn, RA, lupus nephritis, Goodpasturesyndrome, or asthma), as defined herein, means evaluating the subjectfor a change in a disease parameter, e.g., an improvement in one or moresymptoms of a given disorder. The symptoms of many of the abovedisorders (e.g., complement-associated disorders) are well known in theart of medicine. In some embodiments, the evaluation is performed atleast one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, orat least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or atleast 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more,after an administration of a composition described herein. The subjectcan be evaluated in one or more of the following periods: prior tobeginning of treatment; during the treatment; or after one or moreelements of the treatment have been administered. Evaluation can includeevaluating the need for further treatment, e.g., evaluating whether adosage, frequency of administration, or duration of treatment should bealtered. It can also include evaluating the need to add or drop aselected therapeutic modality, e.g., adding or dropping any of thetreatments for a complement-associated disorder described herein.

The compound that reduces serum C5 concentration can be administered toa human in an amount and with a frequency sufficient to reduce serum C5concentration. In the context of reduced serum C5 concentration, atherapeutically effective amount of a C5 antagonist is administered tothe human, e.g., in an amount and with a frequency sufficient tomaintain serum complement activity below 20% of the level of complementactivity in a human in the absence of the C5 antagonist or any othercomplement antagonist. In some embodiments, the compound and C5antagonist can be administered at the same time.

The following examples are intended to illustrate, not limit, theinvention.

Examples Example 1 Treatment of a Human Afflicted with PNH

A human patient is identified by a medical practitioner as having PNH.The patient is one treated with an anti-C5 antibody under the followingdose schedule: (i) 600 mg each week for four weeks; (ii) 900 mg 1 weeklater; and (iii) 900 mg on a biweekly basis thereafter. The patient isthen placed on a new therapeutic regimen. A human C5-specific siRNA ischronically administered to the patient to reduce the concentration ofC5 in the serum of the patient. In the context of reduced serum C5levels, the anti-C5 antibody is administered to the patient at a dose of600 mg every three months. The patient and medical practitioner continueto observe that the patient continues to do as well under the newtherapeutic regimen as under the previous regimen or may even observe asubstantial improvement in PNH symptoms and pathology under the newregimen.

Example 2 Impact of C5 on the Clearance of an Anti-C5 Antibody

To understand the potential impact of antigen-mediated clearance on theoverall clearance rate of an anti-human C5 (hC5) antibody, the followingexperiments were performed using the human neonatal Fc receptor (hFcRn)transgenic mouse model (the mice lack endogenous FcRn but are transgenicfor hFcRn (B6.Cg-Fcgrt^(tm1Dcr) Tg(FCGRT)32Dcr/DcrJ; Stock Number014565, Jackson Laboratories, Bar Harbor, Me.)). The transgenic hFcRnmodel has been described in, e.g., Petkova et al. (2006) Int Immunology18(12):1759-1769; Oiao et al. (2008) Proc Natl Acad Sci USA105(27):9337-9342; and Roopenian et al. (2010) Methods Mol Biol602:93-104.

A single dose of 50 μg of the anti-hC5 antibody in 200 μL of phosphatebuffered saline (PBS) was administered by intravenous (i.v.) injectionto each of six hFcRn transgenic mice. Alternatively, the anti-hC5antibody was pre-incubated in a 4:1 or 2:1 molar ratio of human C5(Complement Technology Inc., Catalog Number: A120) to antibody at 4° C.overnight and the mixtures were administered to the animals. Yet anothergroup of mice were treated with the 4:1 hC5/anti-hC5 antibody mixture onday 0 (the first day of the experiment) followed by an additional 250 μgof hC5 administered by intravenous administration on day 1.

Blood samples of approximately 100 μL were collected from each of themice at days one, three, seven, 14, 21, 28, and 35 following theadministration. The concentration of the anti-hC5 antibody in serum wasmeasured by ELISA.

Antibody serum half-life was calculated using the following formula:

${Halflife} = {T \times \frac{\ln \; 2}{\ln \; \frac{A_{0}}{A_{t}}}}$

where: T=Time elapsed, A_(o)=Original serum concentration of theantibody (concentration at day 1 in the present study) and A_(t)=Amountof the antibody remaining after elapsed time T (minimal detectableconcentration or the last bleeding time point (day 35) in the presentstudy).

The results of the experiment are as follows. In the absence of hC5, thehalf-life of the anti-hC5 antibody in the hFcRn mouse model was13.49±0.93 days. The half-life of the anti-hC5 antibody mixed with a2-fold molar excess of hC5 was 9.58±1.24 days. Mixing the anti-hC5antibody with a 4-fold molar excess of hC5 resulted in an antibodyhalf-life of 5.77±1.86 days. The additional subsequent dose of hC5 tomice administered the 4:1 hC5/anti-hC5 antibody mixture reduced thehalf-life of the antibody to 4.55±1.02 days.

These results indicate that the presence of higher amounts of hC5markedly shorten the half-life of an anti-hC5 antibody. The half-life ofthe antibody depends significantly on the amount of free antibody incirculation. The results suggest that a reduced concentration of humanC5 would significantly reduce the impact of antigen-mediated antibodyclearance and thus increase the half-life of an anti-hC5 antibody in ahuman.

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the disclosure.

1-102. (canceled)
 103. A method for increasing the half-life of aanti-C5 antibody or an antigen-binding fragment thereof, in the serum ofa human, the method comprising: (i) administering to the human acompound that reduces the concentration of complement component C5 inthe serum of the human to thereby reduce the C5 concentration in theserum of the human; and (ii) administering to the human the anti-C5antibody or an antigen-binding fragment thereof, wherein a reduced C5concentration in the serum of the human increases the serum half-life ofthe C5 antagonist administered to the human.
 104. The method of claim103, wherein the human has, is suspected of having, or is at risk fordeveloping, a complement-associated disorder.
 105. A method fordecreasing the frequency at which a therapeutically effective amount ofa anti-C5 antibody or an antigen-binding fragment thereof, must beadministered to a human having, suspected of having, or at risk fordeveloping, a complement-associated disorder, the method comprising: (i)administering to the human a compound that reduces the concentration ofcomplement component C5 in the serum of the human to thereby reduce theC5 concentration in the serum of the human; and (ii) administering tothe human a therapeutically effective amount of the anti-C5 antibody oran antigen-binding fragment thereof, wherein a reduced C5 concentrationin the serum of the human decreases the frequency at which atherapeutically effective amount of a C5 antagonist must be administeredto the human.
 106. A method for decreasing the dosage of a anti-C5antibody or an antigen-binding fragment thereof, required fortherapeutic efficacy in a human having, suspected of having, or at riskfor developing, a complement-associated disorder, the method comprising:(i) administering to the human a compound that reduces the concentrationof complement component C5 in the serum of the human to thereby reducethe C5 concentration in the serum of the human; and (ii) administeringto the human a therapeutically effective amount of the anti-C5 antibodyor an antigen-binding fragment thereof, wherein a reduced C5concentration in the serum of the human decreases the dosage of aanti-C5 antibody required for therapeutic efficacy in the human. 107.The method according to claim 103, wherein the compound: a) reduces thelevel of expression of complement component C5 by one or more cells inthe human; b) inhibits transcription of a human complement component C5gene; c) inhibits translation of an mRNA encoding human complementcomponent C5; d) reduces the stability of an mRNA encoding humancomplement component C5; and/or e) reduces the serum concentration of C5by at least 10%;
 108. The method according to claim 107, wherein thecompound is an siRNA specific for mRNA encoding human complementcomponent C5.
 109. The method according to claim 107, wherein thecompound is an antisense nucleic acid complementary to a mRNA encodinghuman complement component C5.
 110. The method according to claim 103,wherein the anti-C5 antibody or an antigen-binding fragment thereof,binds to complement component C5 and inhibits the cleavage of C5 intofragments C5a and C5b.
 111. The method according to claim 110, whereinthe anti-C5 antibody or antigen-binding fragment thereof is selectedfrom the group consisting of a bispecific antibody, a DVD-Ig antibody, apolyclonal antibody, a recombinant antibody, a diabody, a chimerized orchimeric antibody, a deimmunized antibody, a fully human antibody, asingle chain antibody, a domain antibody, an Fv fragment, an Fdfragment, an Fab fragment, an Fab′ fragment, and an F(ab′)₂ fragment.112. The method according to claim 110, wherein the antibody iseculizumab.
 113. The method according to claim 110, wherein theantigen-binding fragment is pexelizumab.
 114. The method according toclaim 103, wherein the compound is chronically administered to thehuman: a) at least once weekly for at least three weeks; b) at leastonce weekly for at least six weeks; or c) at least once weekly for atleast six months
 115. The method according to claim 103, wherein theanti-C5 antibody or an antigen-binding fragment thereof, is chronicallyadministered to the human.
 116. The method according to claim 103,wherein the anti-C5 antibody or an antigen-binding fragment thereof, andthe compound are both chronically administered to the human.
 117. Themethod according to claim 103, wherein the anti-C5 antibody or anantigen-binding fragment thereof, and the compound are administered tothe human using different routes of administration and/or differentdosing schedules.
 118. The method according to claim 103, wherein theserum half-life of the C5 antagonist is increased 2-fold, 5-fold, or10-fold as compared to the half-life in the absence of administering thecompound.
 119. The method according to claim 103, wherein thecomplement-associated disorder is selected from the group consisting ofparoxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic-uremicsyndrome (aHUS), shiga toxin E. coli-related hemolytic uremic syndrome(STEC-HUS), dense deposit disease (DDD), C3 nephropathy, myastheniagravis, neuromyelitis optica, cold agglutinin disease (CAD),antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV),asthma, age-related macular degeneration (AMD), transplant rejection,Goodpasture's syndrome, glomerulonephritis, vasculitis, rheumatoidarthritis, dermatitis, systemic lupus erythematosus (SLE),Guillain-Barré syndrome (GBS), dermatomyositis, psoriasis, Graves'disease, Hashimoto's thyroiditis, type I diabetes, pemphigus, autoimmunehemolytic anemia (AIHA), idiopathic thrombocytopenic purpura (ITP),lupus nephritis, ischemia-reperfusion injury, thromboticthrombocytopenic purpura (TTP), Pauci-immune vasculitis, epidermolysisbullosa, multiple sclerosis, spontaneous fetal loss, recurrent fetalloss, traumatic brain injury, injury resulting from myocardialinfarction, cardiopulmonary bypass and hemodialysis, and hemolysis,elevated liver enzymes, and low platelets (HELLP) syndrome.
 120. Amethod for treating a human afflicted with a complement-associateddisorder, the method comprising administering to the human atherapeutically-effective amount of an an anti-C5 antibody or anantigen-binding fragment thereof, to complement component C5, whereinthe human has a reduced serum concentration of C5, relative to thenormal serum concentration of C5, as the result of the prioradministration to the patient of a compound that reduces the serumconcentration of C5.
 121. A method for treating a human afflicted with acomplement-associated disorder, the method comprising administering tothe human a compound that reduces the serum concentration of complementcomponent C5, wherein the human is also to be treated with an anti-C5antibody or an antigen-binding fragment thereof.